RNA interference (RNAi) is a specific post-transcriptional gene silencing pathway mediated by intracellular dsRNAs of 21-23 bp in length known as small interfering RNA (siRNA). Since the recent discovery of effective siRNA-mediated gene silencing in mammalian cells, there has been significant validation and enormous interest in the approach from both academic and corporate researchers, for both discovery and therapeutic applications. Despite its great utility, the practice of RNAi would significantly benefit from (i) improved oligonucleotide chemistry for stability and efficacy at low concentrations to limit off-target effects, (ii) efficient and non-toxic delivery methods into cells, and (iii) lower cost siRNA reagents. This proposal describes an interdisciplinary effort between experienced groups to evaluate novel approaches to address these needs. A first-generation bead-based combinatorial thio-modified siRNA library will be generated and screened via cell-based high-throughput high-content screening (HT-HCS) using a novel HT cell imaging platform. Preliminary data within support the use of these oligonucleotide chemistries for RNAi applications and the HT-HCS capabilities needed to scale up to library screening efforts. Hits will be migrated to leads based on confirmatory screening and secondary assays. A focused second-generation combinatorial library will then be generated and screened based on first-generation results, again resulting in hits to be migrated to leads based on follow- up secondary assays. Results from these Phase I studies will be confirmed during Phase II studies which focus on additional target genes and cell lines, including influenza and host immunomodulatory genes with utility in infectious disease research and treatment. Success in this program will have broad impact in the field of RNAi and infectious diseases, resulting in more effective siRNA chemistries available with lower cost of production, HT-HCS cell-based screening methods for study of important models, and the potential for improved in vitro discovery and in vivo therapeutic RNAi approaches. siRNA represents an important new methodology that has led to new drug discovery research approaches, and holds significant promise for direct therapeutic applications. This proposal aims to improve the stability and utility of siRNA molecules and reduce their cost, thereby impacting the study and treatment potential for numerous diseases. [unreadable] [unreadable] [unreadable]